Mutant E. coli strain with increased succinic acid production

ABSTRACT

The invention relates to a mutant strain of bacteria, which either lacks or contains mutant genes for several key metabolic enzymes, and which produces high amounts of succinic acid under anaerobic conditions.

PRIOR RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.11/214,309, filed Aug. 29, 2005, now U.S. Pat. No. 7,223,567, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/604,922filed Aug. 27, 2004, entitled “Mutant E. coli Strain with IncreasedSuccinic Acid Production,” which is incorporated herein in its entirety.

FEDERALLY SPONSORED RESEARCH STATEMENT

The present invention was developed with funds from the National ScienceFoundation. Therefore, the United States Government may have certainrights in the invention.

REFERENCE TO MICROFICHE APPENDIX

Not applicable.

REFERENCE TO A SEQUENCE LISTING

Not applicable.

FIELD OF THE INVENTION

The invention relates to methods of producing succinic acid, malic acid,fumaric acid, and other carboxylic acids in metabolically engineeredmicroorganisms.

BACKGROUND OF THE INVENTION

The valuable specialty chemical succinate and its derivatives haveextensive industrial applications. Succinic acid is used as a rawmaterial for food, medicine, plastics, cosmetics, and textiles, as wellas in plating and waste-gas scrubbing (61). Succinic acid can serve as afeedstock for such plastic precursors as 1,4-butanediol (BDO),tetrahydrofuran, and gamma-butyrolactone. Further, succinic acid and BDOcan be used as monomers for polyesters. If the cost of succinate can bereduced, it will become more useful as an intermediary feedstock forproducing other bulk chemicals (47). Along with succinic acid, other4-carbon dicarboxylic acids such as malic acid and fumaric acid alsohave feedstock potential.

The production of succinate, malate, and fumarate from glucose, xylose,sorbitol, and other “green” renewable feedstocks (in this case throughfermentation processes) is an avenue to supplant the more energyintensive methods of deriving such acids from nonrenewable sources.Succinate is an intermediate for anaerobic fermentations bypropionate-producing bacteria but those processes result in low yieldsand concentrations. It has long been known that mixtures of acids areproduced from E. coli fermentation. However, for each mole of glucosefermented, only 1.2 moles of formic acid, 0.1-0.2 moles of lactic acid,and 0.3-0.4 moles of succinic acid are produced. As such, efforts toproduce carboxylic acids fermentatively have resulted in relativelylarge amounts of growth substrates, such as glucose, not being convertedto desired product.

Numerous attempts have been made to metabolically engineer the anaerobiccentral metabolic pathway of E. coli to increase succinate yield andproductivity (7, 8, 12, 14, 15, 20, 24, 32, 44, 48). Genetic engineeringcoupled with optimization of production conditions have also been shownto increase succinate production. An example is the growth of asuccinate producing mutant E. coli strain using dual phase fermentationproduction mode which comprises an initial aerobic growth phase followedby an anaerobic production phase or/and by changing the headspaceconditions of the anaerobic fermentation using carbon dioxide, hydrogenor a mixture of both gases (35, 49).

Specifically, manipulating enzyme levels through the amplification,addition, or reduction of a particular pathway can result in high yieldsof a desired product. Various genetic improvements for succinic acidproduction under anaerobic conditions have been described that utilizethe mixed-acid fermentation pathways of E. coli. One example is theoverexpression of phosphoenolpyruvate carboxylase (pepc) from E. coli(34). In another example, the conversion of fumarate to succinate wasimproved by overexpressing native fumarate reductase (frd) in E. coil(17, 53). Certain enzymes are not indigenous in E. coli, but canpotentially help increase succinate production. By introducing pyruvatecarboxylase (pyc) from Rhizobium etli into E. coli, succinate productionwas enhanced (14, 15, 16). Other metabolic engineering strategiesinclude inactivating competing pathways of succinate. When malic enzymewas overexpressed in a host with inactivated pyruvate formate lyase(pfl) and lactate dehydrogenase (ldh) genes, succinate became the majorfermentation product (44, 20). An inactive glucose phosphotransferasesystem (ptsG) in the same mutant strain (pfl- and idh-) had also beenshown to yield higher succinate production in E. coli and improve growth(8).

The maximum theoretical yield (molar basis) of succinate from glucoseunder anaerobic conditions is limited to 1 mol/mol, assuming that allthe carbon flux will go through the native succinate fermentativepathway (FIG. 1). The fermentative pathway converts oxaloacetate (OAA)to malate, fumarate and then succinate and this pathway requires 2 molesof NADH per mole of succinate produced. One major obstacle to highsuccinate yield through the fermentative pathway is due to NADHlimitation. This is because one mole of glucose can provide only twomoles of NADH through the glycolytic pathway; however, the formation ofone mole of succinate through the native fermentative pathway requirestwo moles of NADH. Anaerobic production of succinate is also hampered bythe limitations of slow cell growth and production.

Metabolic engineering has the potential to considerably improve processproductivity by manipulating the throughput of metabolic pathways.Specifically, manipulating enzyme levels through the amplification,addition, or deletion of a particular pathway can result in high yieldsof a desired product. What is needed in the art is an improved bacterialstrain that produces higher levels of succinate and other carboxylicacids than heretofore provided.

SUMMARY OF THE INVENTION

Bacteria with more than two pathway proteins inactivated to improvecarboxylic acid production under anaerobic conditions are describedwherein the carboxylic acid produced is succinate, fumarate, malate,oxaloacetate, or glyoxylate. In one embodiment of the invention, theproteins ADHE, LDHA, ACKA, PTA, ICLR, and ARCA are inactivated. Inanother embodiment of the invention various combinations of proteins areinactivated including ADHE, LDHA, ACKA, PTA, ICLR, and ARCA.Inactivation of these proteins can also be combined with theoverexpression of ACEA, ACEB, ACEK, PEPC, PYC, or CITZ to furtherincrease succinate yield.

In one embodiment of the invention, disruption strains are createdwherein the adhE, ldhA, iclR, arcA, and ack-pta genes are disrupted. Inanother embodiment of the invention various combinations of the adhE,ldhA, iclR, arcA, and ack-pta genes are disrupted. The mutant strainsdesignated SBS330MG, SBS440MG, SBS550MG, SBS660MG, and SBS990MG, providesome embodiments of the invention.

Further, an anaerobic method of producing carboxylic acids with a mutantbacterial strain is described, wherein said method comprises inoculatinga culture with a mutant bacterial strain described above, culturing saidbacterial strain under anaerobic conditions, and isolating carboxylicacids from the media. Bacteria strains can be cultured in a flask, abioreactor, a fed batch bioreactor, or a chemostat bioreactor to obtaincarboxylic acids. Carboxylic acid yield can be further increased byculturing the cells under aerobic conditions before succinate productionunder anaerobic conditions.

Additionally, modifying the glycolytic flux in bacteria to distributecarboxylic acid through the OAA-citrate and OAA-malate pathway isdemonstrated. The glycolytic flux can be a ratio between 10-40% citrateand between 90-60% malate, more preferably about 30% citrate and about70% malate. The bacterial strains described above partition glycolyticflux in this manner.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings which are incorporated in and constitute apart of this specification exemplify the invention and together with thedescription, serve to explain the principles of the invention.

FIG. 1 Genetically Engineered Anaerobic Metabolic Pathways. NADHcompeting pathways: lactate (LDH), ethanol (ADH), and acetate pathway(ACK-PTA). The opening of the glyoxylate bypass (ICLR knockout) and theoverexpression of a heterologous pyruvate carboxylase (PYC) from L.lactis. The genetically engineered strain depicted here representsstrain SBS550MG.

FIG. 2 Glucose Consumption and Production Concentration of VariousStrains.

FIG. 3 Product Yield in Various Strains.

FIG. 4 Cumulative Succinate Yield in Mutant Strains.

FIG. 5 Fed Batch Production with strain SBS550MG (pHL413). Solid symbolscorrespond to the metabolites measured on a first run and open symbolscorrespond to the metabolites measured on a replicate run. Glucose wasadded in pulses when glucose dropped to about 20 mM as indicated by thearrows.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Carboxylic acids described herein can be a salt, acid, base, orderivative depending on structure, pH, and ions present. For example,the terms “succinate” and “succinic acid” are used interchangeablyherein. Succinic acid is also called butanedioic acid (C₄H₆O₄).Chemicals used herein include formate, glyoxylate, lactate, malate,oxaloacetate (OAA), phosphoenolpyruvate (PEP), and pyruvate. Bacterialmetabolic pathways including the Krebs cycle (also called citric acid,tricarboxylic acid, or TCA cycle) can be found in Principles ofBiochemistry, by Lehninger as well as other biochemistry texts.

The terms “operably associated” or “operably linked,” as used herein,refer to functionally coupled nucleic acid sequences.

“Reduced activity” or “inactivation” is defined herein to be at least a75% reduction in protein activity, as compared with an appropriatecontrol species. Preferably, at least 80, 85, 90, 95% reduction inactivity is attained, and in the most preferred embodiment, the activityis eliminated (100%). Proteins can be inactivated with inhibitors, bymutation, or by suppression of expression or translation, and the like.

“Overexpression” or “overexpressed” is defined herein to be at least150% of protein activity as compared with an appropriate controlspecies. Overexpression can be achieved by mutating the protein toproduce a more active form or a form that is resistant to inhibition, byremoving inhibitors, or adding activators, and the like. Overexpressioncan also be achieved by removing repressors, adding multiple copies ofthe gene to the cell, or upregulating the endogenous gene, and the like.

The terms “disruption” and “disruption strains,” as used herein, referto cell strains in which the native gene or promoter is mutated,deleted, interrupted, or down regulated in such a way as to decrease theactivity of the gene. A gene can be completely (100%) reduced byknockout or removal of the entire genomic DNA sequence. Use of a frameshift mutation, early stop codon, point mutations of critical residues,or deletions or insertions, and the like, can completely inactivate(100%) gene product by completely preventing transcription and/ortranslation of active protein.

As used herein “recombinant” is relating to, derived from, or containinggenetically engineered material.

Genes are abbreviated as follows: isocitrate lyase (aceA a.k.a. icl);malate synthase (aceB); thy glyoxylate shunt operon (aceBAK); isocitratedehydrogenase kinase/phosphorylase (aceK); acetatekinase-phosphotransacetylase (acKA-pta); alcohol dehydrogenase (adhE);aerobic respiratory control regulator A and B (arcAB); peroxidesensitivity (arg-lac); alcohol acetyltransferases 1 and 2 (atf1 andatf2); putative cadaverine/lysine antiporter (cadR); citrate synthase(citZ); fatty acid degradation regulon (fadR); fumarate reductase (frd);fructose regulon (fruR); fumarase A, B, or C (fumABC); isocitratedehydrogenase (icd); isocitrate lyase (icl); aceBAK operon repressor(iclR); lactate dehydrogenase (ldhA); malate dehydrogenase (mdh);phosphoenol pyruvate carboxylase (pepC); pyruvate formate lyase (pfl);pyruvate oxidase (poxB); phosphotransferase system genes F and G (ptsFand ptsG); pyruvate carboxylase (pyc); guanosine 3′,5′-bispyrophosphatesynthetase I (relA1); ribosomal protein S12 (rpsL); and succinatedehydrogenase (sdh). Δlac(arg-lac)205(U169) is a chromosomal deletion ofthe arg-lac region that carries a gene or genes that sensitizes cells toH₂O₂ (51). PYC can be derived from various species, Lactococcus lactispyc is expressed as one example (AF068759).

Abbreviations: ampicillin (Ap); oxacillin (Ox); carbenicillin (Cn);chloramphenicol (Cm); kanamycin (Km); streptomycin (Sm); tetracycline(Tc); nalidixic acid (Nal); erythromycin (Em); ampicillin resistance(Ap^(R)); thiamphenicol/chloramphenicol resistance (Thi^(R)/Cm^(R));macrolide, lincosamide and streptogramin A resistance (MLS^(R));streptomycin resistance (Sm^(R)); kanamycin resistance (Km^(R));Gram-negative origin of replication (ColE1); and Gram-positive origin ofreplication (OriII). Common restriction enzymes and restriction sitescan be found at NEB® (NEW ENGLAND BIOLABS®, www.neb.com) and INVITROGEN®(www.invitrogen.com). ATCC®, AMERICAN TYPE CULTURE COLLECTION™(www.atcc.org).

Plasmids and strains used in certain embodiments of the invention areset forth in Tables 1 and 2. MG1655 is a F⁻λ⁻ spontaneous mutantdeficient in F conjugation and as reported by Guyer, et al. (18).Pathway deletions were performed using P1 phage transduction and theone-step inactivation based on λ red recombinase (10). The constructionof plasmids and mutant E. coli strains were performed using standardbiochemistry techniques referenced herein and described in Sambrook (38)and Ausebel (5).

TABLE 1 PLASMIDS Plasmid Genotype Ref pTrc99A Cloning vector Ap^(R) 1pDHC29 Cloning vector Cm^(R) 37 pDHK29 Cloning vector Km^(R) 37 pUC19Cloning vector Ap^(R) 60 pHL413 L. lactis pyc in pTrc99A, Ap^(R) 40pCPYC1 L. lactis pyc Cm^(R) 54 pHL531 NADH insensitive citZ in pDHK29,Km^(R) 41 pLOI2514 B. subtilis citZ in pCR2.1-TOPO Km^(R)/Ap^(R) 46

TABLE 2 STRAINS Strain Genotype Ref ATCC # GJT001 MC4100(ATC35695) cadRmutant 45 Δlac(arg-lac) U169rpsL150relA1ptsFSm^(R) MG1655 Wild type(F⁻λ⁻) 18 47076 ™ MG1655 arcA ΔarcA::Km^(R) 23 CD55K ΔldhA::Km^(R) 11YBS121 Δack-pta::Cm^(R) 56 HL5K ΔiclR::Km^(R) 28 SBS100MG ΔadhE, Km^(R)41 SBS110MG ΔadhEΔldhA, Km^(S) 40 SBS330MG ΔadhEΔldhAΔiclR, Km^(S) 41SBS440MG ΔadhEΔldhAΔiclRΔarcA, Km^(S) 41 SBS990MGCΔadhEΔldhAΔack-pta::Cm^(R) 41 SBS550MG ΔadhEΔldhAΔiclRΔack-pta::Cm^(R)41 SBS660MGC ΔadhEΔldhAΔiclRΔarcA- 41 Δack-pta::Cm^(R)

For each experiment the strains were freshly transformed with plasmid ifappropriate. A single colony was restreaked on a plate containing theappropriate antibiotics. A single colony was transferred into a 250 mlshake flask containing 50 ml of LB medium with appropriate antibioticsand grown aerobically at 37° C. with shaking at 250 rpm for 12 hours.Cells were washed twice with LB medium and inoculated at 1% v/v into 2 Lshake flasks containing 400 ml each of LB medium with appropriateantibiotic concentration and grown aerobically at 37° C. with shaking at250 rpm for 12 hours. Appropriate cell biomass (˜1.4 gCDW) was harvestedby centrifugation and the supernatant discarded. The cells wereresuspended in 60 ml of anaerobic LB medium (LB broth mediumsupplemented with 20 g/L of glucose, 1 g/L of NaHCO3) and inoculatedimmediately into the reactor to a concentration of approximately 10OD₆₀₀.

Fermentations were conducted under fully anaerobic conditions. A 1 Lbioreactor with an initial volume of LB medium of 0.66 L withappropriate antibiotics. At time zero, the reactor was inoculated to anOD600 of approximately 10 as described. The initial concentrations ofglucose, antibiotics and cell density in the reactor described are theconcentrations after accounting for the dilution by the inoculum whenthe inoculum is sufficient to change culture concentrations. The pH wasmaintained at 7.0 with 1.0 M Na₂CO₃ and a constant flow rate of 0.2L/min CO₂ was sparged through the culture during the fermentationperiod. The temperature and agitation were maintained at 37° C. and 250rpm respectively. The samples for determination of glucose and productconcentrations were withdrawn from the reactor and filtered through a0.2 μm filter and immediately analyzed by HPLC.

Example 1 Removal of NADH Competing Pathways

Alcohol dehydrogenase (adhE) and lactate dehydrogenase (ldhA) activitywas reduced in SBS110MG to address the loss of NADH to the production ofalcohol and lactate at the expense of succinate (39). SBS110MG (pTrc99A)consumed 11% of the initial glucose with low succinate yield and highacetate yields.

Example 2 Expression of Pyruvate Carboxylase

Expression of the heterologous PYC from Lactococcus lactis (plasmidpHL413) helps to increase the OAA pool by the direct conversion ofpyruvate into OAA, which serves as a precursor of the glyoxylate andfermentative pathway, both of which lead to succinic acid production asdepicted in FIG. 1. Strain SBS990MG showed lower ICL and MS activitiesthan SBS330MG (pHL413) and SBS550MG (pHL413) as expected due to anactive iclR, however, the basal enzyme levels seem to be sufficient todrive the glyoxylate pathway.

The heterologous expression of PYC in succinate producing E. colistrains increases the flux from pyruvate to OAA. PYC diverts pyruvatetoward OAA to favor succinate generation. SBS110MG harboring plasmidpHL413, which encodes the heterologous pyruvate carboxylase from L.lactis, produced 15.6 g/L (132 mM) of succinate from 18.7 g/L (104 mM)of glucose after 24 h of culture in an atmosphere of CO₂ yielding 1.3mol of succinate per mole of glucose. The effectiveness of increasingOAA to increase succinate production was demonstrated.

Example 3 Activating the Glyoxylate Shunt

The inactivation of ICLR diverts acetyl CoA towards the production ofsuccinic acid by activation of the aceBAK operon thus decreasing thecarbon flux through acetate and continuing the recycling of acetyl CoA.In strain SBS330MG (pHL413) ACK-PTA is active and therefore little flowof carbon is diverted to the glyoxylate even when this pathway had beengenetically activated. The activity of the glyoxylate pathway isevidenced by the higher enzyme activity showed by this strain withrespect to the wild type control (data not shown). The strains withhigher ICL and MS activities were SBS330MG (pHL413) and SBS550MG(pHL413). These strains showed more than twice the activity of the wildtype strain. Strain SBS330MG was created as an intermediate strain byinactivating the iclR gene in adh, ldh mutant, SBS110MG. In an ldhA adhEmutant acetyl-CoA is diverted towards the glyoxylate pathway thereforereducing acetate excretion. Even when the glyoxylate pathway has beenconstitutively activated in this strain, acetyl-CoA is channeled throughthe acetate pathway.

As seen in FIG. 3, the SBS330MG strain produced 1.1 mol/mol succinateper glucose, 0.66 mol/mol formate per glucose, and 0.89 mol/mol acetateper glucose. Although SBS330MG(pHL413) produced significant succinate,it also produced the highest levels of formate and acetate.

Example 4 Removal of Aerobic Respiratory Control

The ARCA protein belongs to the two-component (ARCB-ARCA)signal-transduction systems family, and in concert with its cognatesensory kinase ARCB, represents a global regulation system thatnegatively or positively controls the expression of many operons such asseveral dehydrogenases of the flavoprotein class, terminal oxidases,tricarboxylic acid cycle, enzymes of the glyoxylate shunt and enzymes ofthe pathway for fatty acid degradation. Mutant strain SBS440MGC containsa mutant version of arcA, a gene encoding a protein involved in thecontrol of aerobic respiration.

The deletion of arcA in the SBS440MGC strain produced 1.02 mol/molsuccinate per glucose, 0.45 mol/mol formate per glucose, and 0.75mol/mol acetate per glucose, as seen in FIG. 3. The removal of ARCA didnot dramatically affect glucose consumption (FIG. 2), but decreasedoverall succinate yield slightly (FIG. 3).

Example 5 Reducing Acetate Production

In strain SBS990MGC, acetate kinase-phosphotransacetylase (ackA-pta) wasreduced in an alcohol dehydrogenase (adhE) and lactate dehydrogenase(ldhA) background to increase succinate production and reduce NADHconsumption through ethanol and lactate production. Deletion of the ackand pta genes eliminates the major acetate formation pathway. Althoughmutant strain SBS990MG contains adhE, ldhA and ack-pta deletions,SBS990MG still has an intact iclR. Active ICLR may reduce ACEBAKexpression, but residual ACEA and ACEB enzyme activity may still allowresidual glyoxylate shunt activity.

Increased NADH availability in SBS990MG has been implemented to increasesuccinate yield from glucose to 1.6 mol/mol under fully anaerobicconditions (FIGS. 2 and 3). SBS990MG was able to achieve a highsuccinate yield.

Example 6 Dual Succinate Synthesis Route

A pathway design with an activate glyoxylate pathway was previouslydeveloped and examined in silico (9) and implemented in vivo bygenetically engineering E. coli (41) to increase succinate yield andalleviate the NADH availability constraint. Strain SBS550MG (pHL413) wasconstructed to posses a dual succinate synthesis route which divertsrequired quantities of NADH through the traditional fermentative pathwayand maximizes the carbon converted to succinate by balancing the carbonflux through the fermentative pathway and the glyoxylate pathway (whichhas lower NADH requirement) (41). The redesigned pathway showed anexperimental NADH requirement from glucose of ˜1.25 moles of NADH permole of succinate in contrast to the theoretical requirement of 2 molesof NADH per mole of succinate in the wild type E. coli strain.

Inactivation of ack-pta in an ldhA adhE iclR mutant proved to be the keyto channel acetyl-CoA towards the glyoxylate pathway. The deletion ofthe acetate pathway helped conserve carbon molecules in the form ofacetyl-CoA which is diverted to the formation of glyoxylate andsuccinate. Further conversion of glyoxylate to malate to fumarate andfinally to succinate also helped reduce the NADH requirement. Thispathway only requires one mole of NADH to produce two moles of succinatefrom 2 moles of acetyl-CoA and 1 mole of OAA (41).

The performance of strain SBS550MG overexpressing PYC was examined andthe results are shown in FIG. 2. Although strain SBS550MG with PYC cangrow reasonably well under anaerobic conditions, aerobic conditions wereused in this study to accumulate biomass faster before subjecting thecells to the anaerobic succinate production phase. Strain SBS550MG withand without pHL531 consumed 100% of the glucose, and both strainsproduced similar levels of succinic acid (160 mM) from 100 mM glucose.An increase in residual formate and decrease in acetate levels wasobserved with the strain carrying the plasmid pHL531 which overexpressesthe NADH insensitive citrate synthase. The acetate levels found incultures of SBS550MG-based strains were much lower than the acetatelevels found in cultures of SBS110MG (pHL413). Succinate yields ofSBS550MG (pHL413, pHL531) and SBS550MG (pHL413, pDHK29) were verysimilar (about 1.6 mol/mol succinate per glucose).

The fact that the high succinic acid yield of 1.6 mol/mol remainsunchanged despite the overexpression of the NADH insensitive citratesynthase also suggested that the native citrate synthase system isalready sufficient to drive the glyoxylate pathway. In addition, theresults further showed that the dual-route succinate production systemis very robust; the system was able to handle significant perturbationsat the OAA node without significant changes in yield. Apparently thecells are capable of achieving a balanced partition between thefermentative and the glyoxylate pathway in terms of available reducingequivalents and carbon atoms to efficiently maximal succinic acidproduction.

Cultures of the SBS660MG-based strains consumed only 25-35% of theglucose at the end of 24 hours. Both SBS660MG(pHL413, pHL531) andSBS660MG(pHL413, pDHK29) strains produced similar succinate yields ofapproximately 1.7 mol/mol. These yield values are among the highest ofall the strains studied (Table 3). No significant difference wasobserved in the succinic acid, acetate or formate yield of thetransformed mutant strain SBS660MG(pHL413, pHL531) relative to itscontrol (FIG. 3). Deletion of arcA increased succinate production permol glucose, but reduced glucose consumption.

Example 7 Expression of Citrate Synthase

FIG. 1 depicts the various metabolites produced in the E. coli mutantstrains, SBS550MG, SBS660MGC and SBS990MGC transformed with the pyruvatecarboxylase encoding plasmid pHL413 and the citrate synthase expressionplasmid pHL531. The acetate yield in SBS330MG (pHL413) increased withrespect to the control and SBS110MG (pHL413). These results suggest thatthere could be some inhibition of citrate synthase caused by the higherNADH levels and favored by lower NAD+ levels. It is well known that NADHinhibits citrate synthase allosterically (33, 43) and NAD+ is a weakcompetitive inhibitor of NADH binding (13). The absolute in vitroCITRATE SYNTHASE activity observed in SBS330MG (pHL413) was the lowestof all the strains studied.

Strain SBS550MG (pHL413+pDHK29) was used as the control, and SBS550MG(pHL413+pHL531) co-expresses heterologous PYC and citrate synthase.Strain SBS550MG with and without pHL531 consumed 100% of the glucose,and both strains produced similar levels of succinic acid (160 mM) from100 mM glucose. Despite the fact that the acetate pathway was knockedout, low concentrations of acetate were detected in the cultures at theend of the fermentation. An increase in residual formate and decrease inacetate levels was observed with the strain carrying the plasmid pHL531which overexpresses the NADH insensitive citrate synthase.

TABLE 3 CARBOXYLIC ACID YIELD^(A) Strain Succinic acid Formate AcetateSBS550MG (pHL413 + 1.59 ± 0.01 0.13 ± 0.03 0.08 ± 0.01 pDHK29) SBS550MG(pHL413 + 1.61 ± 0.01 0.18 ± 0.04 0.06 ± 0.00 pHL531) SBS660MG (pHL413 +1.72 ± 0.13 0.11 ± 0.01 0.09 ± 0.00 pDHK29) SBS660MG (pHL413 + 1.73 ±0.18 0.10 ± 0.06 0.10 ± 0.03 pHL531) SBS990MG (pHL413 + 1.50 ± 0.00 0.26± 0.03 0.09 ± 0.01 pDHK29) SBS990MG (pHL413 + 1.58 ± 0.01 0.15 ± 0.010.06 ± 0.00 pHL531) ^(A)mol of product/mol of glucose during a 24 hranaerobic production phase

The expression of the NADH citrate synthase in the SBS660MG(pHL413,pHL531) strain increased the glucose consumption as well as themetabolites produced by about 40% when compared with theSBS660MG(pHL413, pDH29) control strain. Both SBS660MG(pHL413, pHL531)and SBS660MG(pHL413, pDHK29) strains produced similar succinate yieldsof approximately 1.7 mol/mol. These yield values are among the highestof all the strains studied (Table 3). The succinic acid level producedby the SBS990MG(pHL413, pDHK29) strain was very similar to that of theSBS550MG(pHL413, pDHK29) strain.

The results showed that the dual-route succinate production system isvery robust; the system was able to handle significant perturbations atthe OAA node without significant changes in yield. The engineered cellsare capable of achieving a balanced partition between the fermentativeand the glyoxylate pathway in terms of available reducing equivalentsand carbon atoms to efficiently maximal succinic acid production. Theseresults further support the hypothesis that there might be some degreeof activation of the glyoxylate pathway when adhE, ldhA and ack-pta areinactivated.

Example 8 NADH Flux Analysis

Measured fluxes obtained under batch cultivation conditions were used toestimate intracellular fluxes and identify critical branch point fluxsplit ratios. The comparison of changes in branch point flux splitratios to the glyoxylate pathway and the fermentative pathway at the OAAnode as a result of different mutations revealed the sensitivity ofsuccinate yield to these manipulations.

A metabolic matrix was constructed based on the law of mass conservationand on the pseudo-steady state hypothesis (PSSH) on the intracellularintermediate metabolites. The stoichiometric matrix obtained from theset of linear equations formulated based on the metabolic network shownin FIG. 1 gave a square matrix of dimensions 15×15 based on solely themeasured metabolites, PSSH balances on the intracellular metabolites andNADH balance. For the NADH balance it was assumed that the NADHproduction and consumption must be equal. Derivation of the redoxbalance does not take into account any carbon assimilated into biomass.The biomass (υ₂) was assumed to be zero for all the mutants except thewild type and was used as a measurement which produced an overdeterminedsystem where the solution was found by least-squares fit. Themathematical representation, stoichiometric matrix and net conversionequations relating the various species in the network are presented inTable 4: Enzymatic Reactions.

TABLE 4 ENZYMATIC REACTIONS Reaction Enzyme(s) 1 Glucose + PEP

Glucose 6-P + Pyruvate Glucose: PTS enzymes; Enzyme I, HPr, II^(Glc),III^(Glc) 2 Glucose-6-P

Biomass 3 Glucose 6-P + ATP

2 Glyceraldehyde 3-P + ADP a. Glucose 6-P

Fructose 6-P Glucose-6-phosphate isomerase b. Fructose-6-P + ATP

Fructose Phosphofructokinase, 1,6-diP + ADP PFK c. Fructose 1,6-diP

Dihydroxyacetone-P + Fructose-diP aldolase Glyceraldehyde 3-P d.Dihydroxyacetone-P

Glyceraldehyde 3-P Triose-P isomerase 4 Glyceraldehyde 3-P + NAD⁺ +P_(i) + 2ADP

Pyruvate + H₂O + NADH + H⁺ + 2ATP a. Glyceraldehyde 3-P + NAD⁺ + 3-PGlyceraldehyde P_(i)

Glycerate 1,3-diP + NADH + H⁺ dehydrogenase b. Glycerate 1,3-diP + 3-Pglycerate kinase ADP

Glycerate 3-P + ATP c. Glycerate 3-P

Glycerate 2-P P glycerate mutase d. Glycerate 2-P

PEP + H₂O Enolase e. PEP + ADP

Pyruvate + ATP Pyruvate kinase 5 I. PEP + CO₂

OAA + P_(i) PEP carboxylase II. Pyr + ATP + CO₂

oxaloacetate + Pyruvate carboxylase ADP + P_(i) 6 Pyruvate + NADH

lactate + NAD⁺ Lactate dehydrogenase 7 Pyruvate + HSCoA

Formate + Acetyl-CoA Pyruvate formate-lyase 8 Formate

CO₂ + H₂ Formate hydrogen lyase 9 Acetyl-CoA + P_(i) + ADP

Acetate + HSCoA + ATP a. Acetyl-CoA + P_(i)

Acetyl-P + HSCoA Acetate phospho- transferase, PTA b. Acetyl-P + ADP

Acetate + ATP Acetate kinase, ACK; or, chemical hydrolysis 10Acetyl-CoA + 2 NADH + 2 H⁺

Ethanol + HSCoA + 2 NAD⁺ a. Acetyl-CoA + NADH + Aldehyde H⁺

Acetaldehyde + HSCoA + NAD⁺ dehydrogenase b. Acetaldehyde + NADH +Alcohol H⁺

Ethanol + NAD⁺ dehydrogenase 11 2 acetyl-CoA + H₂O + oxaloacetate

Malate + Succinate HSCoA + CoA a. acetyl-CoA + H₂O + Citrate synthaseoxaloacetate

citrate + CoA b. citrate

isocitrate Aconitate hydratase c. Isocitrate

glyoxalate + succinate Isocitrate lyase d. glyoxalate + acetylCoA

Malate + HSCoA Malate synthase 12 oxaloactetate + NADH

Fumarate + NAD⁺ + H₂O a. oxaloactetate + NADH

Malate + NAD⁺ Malate dehydrogenase b. malate

fumarate + H₂O Fumarase C 13 Fumarate + NADH + P_(i) + Fumaratereductase ADP

Succinate + NAD⁺ + ATP

Vector r(t) (m×1), where m=15, 16 or 17) denotes the net conversionrates for the various metabolites:r^(T)=[r₁ . . . r₈ 000000000]

For MG1655 (pHL413), the balances for H2 and biomass were excluded,therefore matrix A was reduced to a 15×15 square matrix. The exactsolution was obtained by equation 1:υ(t)=A ⁻¹ r(t)  (1)

For the mutant strains SBS110MG (pHL413), SBS330MG (pHL413), SBS550MG(pHL413) and SBS990MG (pHL413) exclusion of the H2 balance and inclusionof biomass gave as a result an overdetermined system (m=16 and n=15)which was solved using the least squares fit approach and was estimatedaccording to the following equation 2:υ(t)=(A ^(T) A)⁻¹ A ^(T) r(t)  (2)

The estimation of the time varying concentrations of excretedmetabolites was determined between t=0 h and t=6 h or 7 h and based onthe average cell density as follows (4):

$\begin{matrix}{r_{i} = \frac{{C_{i}\left( {t + {\delta\; t}} \right)} - {C_{i}(t)}}{\delta\; t \times \overset{\_}{X}}} & (3) \\{{Where},{\overset{\_}{X} = \frac{{X\left( {t + {\delta\; t}} \right)} - {X(t)}}{\ln\left\{ \frac{X\left( {t + {\delta\; t}} \right)}{X(t)} \right\}}}} & (4)\end{matrix}$

It was found that the succinate yield is relatively more sensitive tothe split ratio at the OAA node than at the PEP/PYR node. The mostfavorable split ratio to obtain the highest succinate yield was thefraction partition of OAA to glyoxylate of 0.32 and 0.68 to thefermentative pathway obtained in strains SBS550MG (pHL413) and SBS990MG(pHL413). The succinate yields achieved in these two strains were 1.6mol/mol and 1.7 mol/mol respectively. It has been shown that an activeglyoxylate pathway in an idhA, adhE, ack-pta mutant strain isresponsible for the high succinate yields achieved anaerobically. Highintracellular levels of NADH and acetyl-CoA compared to the wild typewere found in SBS550MG (pHL413) and SBS990MG (pHL413) at the onset ofthe anaerobic fermentation, but these levels rapidly dropped suggestingthat these strains are capable of handling high glycolytic fluxes inspite of inactivation of enzymes downstream of the PEP/PYR branch point.

The glycolytic fluxes (υ₁ through υ₄) of strain SBS550MG (pHL413)increased with respect to SBS330MG (pHL413) but decreased with respectto the wild type control strain. The PFL flux (υ₇) also decreased withrespect to the wild type control strain causing a major reduction in theacetate and formate flux. These glycolytic fluxes demonstrate that highsuccinate yield is dependent upon partitioning of carboxylic acidproduction between OAA-citrate and OAA-malate. The flux partitioningbalances NADH consumption and leads to increased production ofsuccinate. Partitioning in high succinate producing strains variesbetween 10-40% citrate derived succinate and 90-60% malate derivedsuccinate production (see FIG. 1).

TABLE 5 METABOLIC FLUXES MG1655 SBS110MG SBS330MG SBS550MG SBS990MG FluxTo: (pHL413) (pHL413) (pHL413) (pHL413) (pHL413) ν1 Glucose Uptake* 3.462.78 1.28 2.13 1.96 ν2 Biomass 0.07 0.00 0.00 0.00 0.00 ν3 G-3-P 3.382.83 1.37 2.16 2.08 ν4 PEP + Pyruvate 6.77 5.66 2.77 4.32 4.19 ν5 OAA2.90 2.94 1.31 2.57 2.53 ν6 Lactate* 0.00 0.00 0.21 0.00 0.00 ν7Intracellular Formate 3.87 2.74 1.27 1.76 1.72 ν8 H₂ 0.42 0.27 0.00 0.770.63 ν9 Acetate* 2.58 2.35 1.22 0.14 0.12 ν10 Ethanol* 0.65 0.00 0.000.00 0.00 ν11 Glyoxylate Succinate 0.32 0.21 0.05 0.82 0.83 ν12 Malate2.58 2.74 1.28 1.76 1.72 ν13 Intracellular Succinate 2.90 2.97 1.35 2.582.58 ν_(RF) Residual Formate* 3.45 2.47 1.62 0.99 1.09 ν_(ES) ExcretedSuccinate* 3.22 3.19 1.43 3.41 3.44 CO₂ −2.48 −2.67 −1.31 −1.80 −1.90Succinate Yield 0.93 1.15 1.12 1.60 1.75 (NADH)_(U)/Gl 1.96 2.04 2.172.03 2.14 (NADH)_(s)/Succinate 1.70 1.79 1.83 1.27 1.25

Example 9 Fed Batch Anaerobic Fermentation

A bioreactor generates higher productivity due to a more controlledenvironment. A batch experiment was performed in duplicate to examinethe efficiency of SBS550(pHL314) in producing succinate under controlledconditions (FIG. 5). In this experiment, the bioreactor was initiallycharged with LB and supplemented with 86 mM of glucose. At time zero,the reactor was inoculated to an OD of 17. Glucose was added in pulseswhen glucose dropped to about 20 mM as indicated by the arrows.Differences in initial glucose concentration and in glucose additiontime between runs did not affect strain performance.

As seen in FIG. 5, the strain is very efficient in producing succinicacid. The succinic acid production ranges from 12 to 9 mM ofsuccinate/hr with a very efficient yield of 1.6 mole/mole. The systemonly produced very minute quantities of other metabolites, such asformate and acetate as byproducts. It is also expected that the systemcan be further optimized to improve succinate yield to 1.8, 1.9, or 2.0mole/mole, or even higher.

All of the references cited herein are expressly incorporated byreference. References are listed again here for convenience:

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1. A method of producing succinate comprising: a) generating agenetically engineered Escherichia coli bacterial cell comprising adisruption of: i) lactate dehydrogenase (ldh), ii) alcohol dehydrogenase(adh), iii) aceBAK operon repressor (iclR), and iv) both acetate kinaseand phosphotransacetylase (ack-pta), b) culturing said bacteria underanaerobic conditions in a bioreactor, wherein a glycolytic flux fromoxaloacetate (OAA) is partitioned in a ratio between 10-40% citrate andbetween 90-60% malate, wherein said bacteria produces greater than 1.5moles of succinate per mol of glucose.
 2. The method of claim 1, whereinsaid glycolytic flux is partitioned in a ratio of about 30% citrate andabout 70% malate.
 3. The method of claim 1, wherein said bacteriacomprise an expression construct encoding pyruvate carboxylase (pyc),wherein said pyc is from Lactobacillus lactis.
 4. A method of producingsuccinic acid comprising: a) generating a genetically engineeredEscherichia coli bacterial cell comprising a disruption of: i) ldh, ii)adh, iii) iclR, and iv) ack-pta, b) culturing said bacteria underanaerobic conditions in a fed-batch reactor, wherein a glycolytic fluxfrom oxaloacetate (OAA) is partitioned in a ratio between 10-40% citrateand between 90-60% malate, wherein said bacteria produces greater than1.5 moles of succinic acid per mol of glucose.
 5. The method of claim 4,wherein said glycolytic flux is partitioned in a ratio of about 30%citrate and about 70% malate.
 6. The method of claim 4, wherein saidbacteria comprise an expression construct encoding pyc, wherein said pycis from Lactobacillus lactis.